2,4,6-Trinitrotoluene (TNT), the most widely used explosive, causes serious health and environment problems and is recalcitrant to degradation. Herein, we investigated the metabolic pathway of TNT catalyzed by purified nitroreductases NfsA and NfsB from Escherichia coil, and enhanced the transformation activity by structure-based mutagenesis. The two nitroreductases play similar roles in the transformation of TNT, which is converted initially to 4-hydroxylamino-2,6-dinitrotoluene and then 2,4-dihydroxylamino-6-nitrotoluene without production of any amine products. Of the mutants constructed, F123A and F124W mutants dramatically enhanced the catalytic activity towards TNT and its derivatives, suggesting that these two adjacent phenylalanines displayed different effects on the catalytic activity. Substitutions with Ala or Asn at Phe124 greatly impaired the reduction activity against TNT, while the mutation of Phe to Trp resulted in a substantially reduced apparent K-m and improved activity, implying the importance of aromatic residues at position 124 for substrate binding. In contrast, Phe at position 123 caused adverse effects on the activity, may be due to the steric constraint limiting movement of Phe124. (C) 2015 Elsevier Ltd. All rights reserved.